The eukaryotic cell cycle is tightly regulated by the action of the cyclin-dependent kinases (Cdk/cyclins). The kinase activity of these complexes is directly responsible for the initiation and progression of successive phases of the cell cycle by phosphorylation of proteins required for the activation of numerous structural and regulatory genes. Therefore, reversible regulation of Cdk/cyclin kinase activity is key to controlling the cell cycle. While it is known that the Cdc25 phosphatases perform the final and critical activation of the Cdk/cyclins, the details of their catalytic mechanism of action, substrate specificity, and regulation have remained elusive. Mechanistic analysis of the enzymes involved in cell cycle regulation, Cdc25 in particular, will provide fundamentally new insights and novel approaches to drug discovery in a system that is of great importance in development, cancer and other diseases. The long-term goal of this research project is to understand the kinetic and molecular mechanisms of the regulation of the cell cycle by the Cdc25 phosphatases using a combination of kinetic analyses of highly purified proteins in vitro, computer modeling of protein networks, and determination of phosphorylation levels in vivo. First, the detailed reaction mechanism of the Cdc25 phosphatase with their natural substrates, the Cdk/cyclins will be elucidated. These studies include the determination of the putative catalytic acid in the reaction mechanism, which may lie on the substrate itself and thus provide an elegant example of how nature has solved the problem of controlling potentially promiscuous phosphatase. Also, the residues outside of the active site involved in substrate recognition will be identified and thus potentially guide the development of inhibitors specific to the Cdc25 by targeting regions unique to the Cdc25 and Cdk/cyclin interaction. Further work will analyze the substrate specificity among the three Cd25 homologues and various Cdk/cyclins (Cdk2/CycA vs. Cdc2/CycB) to elucidate the basic parameters of substrate discrimination that prevents undesired crosstalk among the numerous possible enzyme/substrate combinations. Mutagenesis and truncation of Cdc25 proteins as well as the use of chimeric Cdc25 proteins will be used to demonstrate how this specific recognition is achieved and may reveal a general paradigm for substrate recognition by the Cdc25s. Finally, utilizing these purified components, the individual kinetic components of the mutual activation loop, in which the Cdc25 phosphatases activate the Cdk/cyclins that in turn feed back to activate the phosphatases, will be characterized. Kinetic characterization of this two-component loop will provide the basis for quantitative modeling of these complex networks to yield an understanding of cell cycle regulation beyond the concept of simple on/off switches. Thus, this research will greatly expand the knowledge of cell cycle transitions, their control on a molecular basis, and their role in oncogenesis.
Showing the most recent 10 out of 17 publications
